Biotinylated peanut agglutinin pna

Erythrocyte-depleted spleen or bone marrow cells were stained and analyzed as previously described [31 (link)]. Half a million RBC-depleted splenocytes were incubated with mouse IgG (Sigma-Aldrich) for 15 min prior to staining with various combinations of directly-conjugated mAbs. The following directly conjugated mAbs were purchased from BD Biosciences (San Diego, CA): FITC-conjugated anti-CD86 (GL1), -CD95 (Jo2), -CD4 (RM4-5), -CD3 (145-2C11), -CD44 (IM7), -CD62L (MEL-14), -CXCR5 (2G8), and -GL7 (GL7); biotin-conjugated anti-B220 (RA3-6B2), -IgM^a (DS-1), -CD24 (M1/69), -CD23 (B3B4), -CD138 (281–2), -CD19 (1D3) and -CD21 (7G6); allophycocyanin-conjugated anti-IFN-γ (XMG1.2); Alexa Fluor488-conjugated anti-IL17A (TC11-18H10); and PE-anti-IL21 (4A9). Biotinylated peanut agglutinin (PNA) was purchased from Sigma-Aldrich (St. Louis, MI) and biotinylated polyclonal rabbit anti-HEL Ab was purchased from Rockland (Gilbertsville, PA). Isotype controls were purchased from Caltag (Buckingham, UK). Allophycocyanin-, PE-, or PerCP-conjugated streptavidin (BD Biosciences) were used to reveal biotinylated Ab staining. Dead cells were excluded by staining with propidium iodide (PI, Sigma-Aldrich), 0.6 μg/ml. Stained cells were acquired and analyzed using a BD FACSCalibur and FACSDiva software, or using a BD LSRII flow cytometer and FlowJo software (TreeStar, San Carlos, CA).

Rabbit anti-GRASP55 (ref. 10598-1-AP, ProteinTech), rabbit anti-JAM-B 829 [50 (link), 51 (link)], rabbit anti-JAM-C 501 [51 (link)], goat anti-JAM-C (ref. AF1213, R&D system), mouse anti-GM130 (ref. 610822, BD Biosciences), mouse anti-SP56 (ref. MA1-10866, ThermoFisher Scientific), rabbit anti-Nectin3 (ref. ab63931, Abcam), rabbit anti-SYCP3 (ref. ab15093, Abcam) and mouse anti-actin (ref. A3853, Sigma) primary antibodies and biotinylated PeaNut Agglutinin (PNA, ref. L6135, Sigma) were used for immunostaining and immunoblotting. Appropriate anti-rabbit, anti-goat or anti-mouse secondary antibodies were obtained from Jackson Immuno-research Laboratories.

Spleens were harvested and single cell suspensions prepared using 100 µm filters. Flow cytometry for B and T cells was performed as previously described^{35 (link)}. Anti-CD19, anti-B220, anti-CD21, anti-CD23, anti-IgM, anti-CD3, anti-CD4, anti-CD8, anti-IL-17A, anti-IL-4 and anti-IFN-γ antibodies were obtained from eBioscience (San Diego, CA, USA), anti-IgD, anti- CD95, anti-CD138, anti-IgG1 and anti-IgG2ab antibodies from BD BioSciences, anti-IL-10 antibody was purchased from Biolegend (San Diego, CA, USA) and biotinylated peanut agglutinin (PNA) from Sigma-Aldrich (St Louis, USA).
Samples were measured on a FACS Canto II HTS or a LSR II flow cytometer (BD BioSciences) and analysis was performed using FlowJo v7.6 research software (Tree Star Inc. Ashland, OR).